Plant Produced Human Papillomavirus Pseudovirion

Patent metadata

Field	Value
Publication number	US-2016168583-A1
Application number	US-201414907323-A
Country	US
Kind code	A1
Filing date	Jul 25, 2014
Priority date	Jul 25, 2013
Publication date	Jun 16, 2016
Grant date	—

Abstract

The present invention relates to a method of producing HPV pseudovirions in plant cells, the plant produced pseudovirions per se, a neutralisation assay using the plant produced pseudovirions and pharmaceutical compositions comprising the plant produced pseudovirions.

First claim

1 . A method for producing a human papillomavirus (HPV) pseudovirion in a plant cell, the method comprising the steps of:
(i) introducing into the plant cell:
(a) a first nucleic acid encoding a HPV L1 polypeptide; and
(b) a second nucleic acid encoding a HPV L2 polypeptide,
wherein the first and second nucleic acids are contained on at least one expression vector,
(c) a replicating vector comprising a third nucleic acid encoding a heterologous polypeptide;
(ii) expressing the HPV L1 polypeptides and HPV L2 polypeptides in the plant cell, and (iii) replicating the replicating vector in the plant cell, in order to produce a high copy number of the replicating vector in the plant cell, wherein the expressed HPV L1 and HPV L2 polypeptides assemble, together with a copy of the replicating vector and encapsidate the replicating vector to produce a HPV pseudovirion.

2 . The method of claim 1 , wherein the first and second nucleic acids are operably linked to regulatory sequences that allow for expression of the HPV L1 and HPV L2 polypeptides.

3 . The method of claim 1 , wherein replication of the replicating vector is initiated by a regulatory protein.

4 . The method of claim 3 , wherein the regulatory protein is encoded by a fourth nucleic acid operably linked to a regulatory sequence that allows for the expression of the regulatory protein, wherein the fourth nucleic acid is expressed from at least one of the group selected from:
a nucleic acid sequence contained on the replicating vector; (ii) a nucleic acid sequence contained on the at least one expression vector; (iii) a nucleic acid sequence contained on an independent vector, not being the vector of (i) or (ii) above; or (iv) a nucleic acid sequence integrated into the genomic DNA of the plant cell; wherein expression of the regulatory protein in the presence of the replicating vector results in replication of the replicating vector.

5 . The method of claim 1 , wherein the third nucleic acid sequence is operably linked to a regulatory sequence that allows for expression of the heterologous polypeptide in a mammalian cell.

6 . The method of claim 1 , wherein the third nucleic acid encoding the heterologous polypeptide, comprises a gene selected from the group consisting of a reporter gene, a therapeutic gene or a gene encoding an antigenic polypeptide.

7 . The method of claim 6 , wherein the gene encoding a heterologous polypeptide is a reporter gene selected from a luciferase gene or a secreted alkaline phosphatase gene.

8 . The method of claim 1 , further comprising a step of recovering the HPV pseudovirion from the plant cell.

9 . An assay for detecting the presence of a neutralising antibody to HPV in a subject, the assay including the steps of:
(i) combining a HPV pseudovirion produced according to the method of claim 1 , with a biological sample from the subject to form a biological sample composition, wherein the heterologous polypeptide is a reporter polypeptide; and (ii) combining a HPV pseudovirion produced according to the method of claim 1 , with a control sample, wherein the control sample does not contain a HPV neutralising antibody, to form a control sample composition, wherein the heterologous polypeptide is a reporter polypeptide; (iii) contacting and incubating a mammalian cell capable of being infected with HPV with the biological sample composition of (i) or the control sample composition of (ii); (iv) assaying the expression of the reporter polypeptide; wherein decreased expression of the reporter polypeptide in the mammalian cells contacted with the biological sample composition, as compared to mammalian cells contacted with the control sample composition is indicative of the presence of a HPV neutralising antibody in the biological sample.

10 . The assay of claim 9 wherein the reporter polypeptide is selected from either a luciferase or a secreted alkaline phosphatase polypeptide.

11 . The assay of claim 9 wherein the subject is a human.

12 . A HPV pseudovirion comprising a capsid, wherein the capsid comprises a HPV L1 and a HPV L2 polypeptide, wherein the capsid encapsidates a replicating vector encoding a heterologous polypeptide, the heterologous polypeptide being operably linked to a regulatory sequence that allows its expression in a mammalian cell, wherein the HPV pseudovirion is produced in and recovered from a plant cell.

13 . The HPV pseudovirion of claim 12 , wherein replication of the replicating vector may be initiated, in a mammalian cell infected by the HPV pseudovirion, in the presence of a regulatory protein.

14 . The HPV pseudovirion of claim 13 , wherein the regulatory protein is encoded by a nucleic acid sequence operably linked to a regulatory sequence that allows for the expression of the regulatory protein in the mammalian cell, where in the regulatory protein may be expressed from any one of the group consisting of:
(i) a nucleic acid sequence contained on the replicating vector; (ii) a nucleic acid sequence contained on an independent vector; or (iii) a nucleic acid sequence integrated into the genomic DNA of the mammalian cell, wherein expression of the regulatory protein in the mammalian cell results in the replication of the replicating vector.

15 . The HPV pseudovirion of claim 12 , wherein the heterologous polypeptide is selected from the group consisting of a reporter polypeptide, a therapeutic polypeptide or an antigenic polypeptide.

16 . A pharmaceutical composition comprising a HPV pseudovirion produced by the method of claim 1 and a pharmaceutically acceptable carrier or adjuvant.

17 . A pharmaceutical composition comprising the HPV pseudovirion of claim 12 and a pharmaceutically acceptable carrier or adjuvant.

Assignees

• Univ Cape Town

Inventors

• Rybicki Edward P
• Hitzeroth Inga Isabel

Classifications

• A61K35/76

  Viruses; Subviral particles; Bacteriophages · CPC title

• C12N2750/12043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2710/20023

  Virus like particles [VLP] · CPC title

• C12N15/8203 Primary

  Virus mediated transformation · CPC title

• C12N15/8257

  for the production of primary gene products, e.g. pharmaceutical products, interferon · CPC title